1. Field of the Invention
This invention relates to a method for recovering Bacillus thuringiensis crystal protein from an aqueous fermentation culture containing lysed cells, spores and insecticidal crystal protein.
2. Description of the Prior Art
Bacillus thuringiensis (Bt) is a well known bacterium characterized by its ability to produce crystal protein inclusions during its sporulation phase. Many of these specific insecticidal activity. An excellent review, "Insecticidal Crystal Proteins of Bacillus thuringiensis", has been published by Hofte et al. in Microbiol.Rev. (1989) 53:242-252.
Insecticidal compositions containing sporulated Bt, i.e., lysed Bacillus thuringiensis cells, spores, and crystal proteins, in spent fermentation culture used to prepare such Bt's, are sold commercially for insect pest control. Improved formulations, containing only the insecticidal crystal protein in combination with agriculturally-acceptable adjuvants for topical application to the desired plant, soil or other locus, are desirable for several reasons: Concentration levels of the crystal protein, including combinations of crystal proteins, may be precisely controlled. Impurities, spores, Bt cell debris, and spent fermentation media components would be absent from the formulations.
Production of such improved insecticidal formulations requires commercial quantities of crystal protein in relatively pure form. Most of the protein purification techniques described in the literature are laboratory methods for small scale preparation of Bt endotoxin protein. These Bt protein recovery or purification techniques are generally costly, complex, and have low recovery yields.
Density gradient centrifugation has been used for the physical separation of Bt crystal protein from lysed cells, spores, and other solids in a spent fermentation medium, based on the different densities of these components. Density gradient centrifugation techniques are described by Nickerson et al., Europ.J.Appl.Microbiol.Biotechnol. (1981) 13:213-215 (using sodium bromide), and by Milne et al., J.Invert.Path. (1977) 29:230-232 (using Renografin). Density gradient separations generally require the use of expensive materials as the separation medium, and the recovered crystal protein often requires further purification to remove associated impurities not removeable by washing.
Other laboratory techniques for isolation of Bt crystal protein involve solubilization of the crystal protein by various means and purification of the solubilized protein by ion exchange chromatography or gel filtration chromatography. These latter procedures are not ordinarily suitable for large scale, commercial purification of Bt endotoxin protein, primarily because of their high cost and low recovery yields. Aronson et al., Appl.Environ. Microbiol. (1987) 53:416-421, digest Bt proteins from a fermentation powder with trypsin at pH 9 and precipitate the digested protein with ammonium sulfate. After the precipitated protein is redissolved at pH 9.5 in buffered solution, it is purified further using ion exchange and hydrophobic-interaction chromatography. Tyski et al., Biochem.Biophys.Res.Comm. (1986) 141:106-111, extract Bt crystal protein obtained from a fermentation culture with bicarbonate buffer at pH 9 containing a reducing agent. The solubilized protein is precipitated with ammonium sulfate, then resolubilized and proteolytically digested with trypsin in a buffered solution at pH 8. The digested protein is next purified by ion exchange chromatography. Hofte et al., Eur.J.Biochem. (1986) 161:273-280, use either bicarbonate extraction buffer at pH 9.5 or TRIS-HCl-containing and urea-containing buffer at pH 8 (both with protein reducing agent) to solubilize the crystal protein. The solubilized protein is purified further by anion exchange or gel filtration chromatography.
Bulla et al., J.Biol.Chem. (1981) 256:3000-3004, and Biochem.Biophys.Res.Comm. (1979) 91:1123-1130, utilize sodium hydroxide at pH 12 for solubilization of Bt crystal protein obtained by density gradient centrifugation. The solubilized protein is purified further using gel filtration chromatography. Lilley et al., J.Gen.Microbiol. (1980) 118:1-11, dissolve partially-purified protein crystals in an alkaline buffer at pH 10.5 containing a reducing agent, followed by digestion of the solubilized protein with trypsin and other proteolytic enzymes. A trypsin-resistant protein is purified further by gel filtration and ion exchange chromatography.
The present invention provides a straight forward method of purifying Bt crystal protein without resorting to density gradient centrifugation, ion exchange chromatography, gel filtration chromatography, molecular sieve filtration, or other costly separation techniques utilized in the prior art. The method of this invention recovers a purified endotoxin protein in good yields and without significant loss of the protein toxin's insecticidal activity.
In this disclosure, the terms "crystal protein", "crystal" or "crystalline endotoxin protein", "protein endotoxin" or "toxin", and "Bt toxin" are used synonomously to refer to Bt-type endotoxin protein. Such endotoxin protein may be in protoxin form or activated form, unless indicated otherwise.